Benny K. K. Chan

Morphological and genetic differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita

The common intertidal barnacle Tetraclita squamosa occurs in two morphologically and genetically distinct forms in East Asia. The north‐western Pacific form (Japan, Okinawa and Taiwan) has green parietes and the tergo‐scutal flaps are black without any patterns. The south China form (Xiamen, Hong Kong) also has green parietes but the tergo‐scutal flaps are black with two white spots on the tergal and scutal margin. Compared to the NW Pacific form, the south China form has a beaked tergum, a sharper tergal spur and cirrus I lacks serrulate type setae that have four rows of setules. The two forms differ by 15–16% in COI divergence, which is comparable to values for other congeneric barnacle species. The 12S rRNA and ITS1 sequences are also distinct between the two forms. Our results support the conclusion that the two forms are genetically differentiated species. We describe the NW Pacific form as a new species, Tetraclita pacifica. We are treating the other species as Tetraclita squamosa based on the fact that Pilsbry, in 1916, redescribed T. squamosa squamosa using samples collected from south China and the Philippines. Further studies are needed to confirm the identity and geographical distribution of the ‘widely distributed’T. squamosa in the Indo‐West Pacific.

Host-associated speciation in the coral barnacle Wanella milleporae (Cirripedia: Pyrgomatidae) inhabiting the Millepora coral.

Speciation by host shift is a common phenomenon observed in many symbiotic animals. The symbiont–host interaction is highly dynamic, but it is poorly documented in the marine realm. In the present study, we examined the genetic and morphological differentiation of the coral barnacle Wanella milleporae (obligate to fire corals) collected from four different Millepora host species in Taiwan to investigate the host specificity of this barnacle. Phylogenetic analysis of mitochondrial COI gene for 241 individuals of Wanella revealed five distinct clades, whose sequence divergences are comparable to values between other cogeneric barnacle species. The five clades also differ in shell and opercular plate morphology and colour. Genetic and morphological differentiations together strongly suggest the presence of cryptic species. Although the five clades do not display species‐level host specificity, they showed a significant difference in preference on host growth form. Clades 1 and 2 were predominantly found on encrusting Millepora exaesa and Millepora platyphylla, while clades 3, 4 and 5 live exclusively on branching‐form fire corals Millepora dichotoma and Millepora tenella. Phylogeny inferred from the combined mitochondrial COI, 16S and 12S (2182 bp) analysis suggests the division of the five clades into two major lineages congruent with the morphology of the host coral. Multiple independent invasions to the same form of host and subsequent speciation are evident in the Red Sea and Taiwan. Our results indicate that ecological/sympatric speciation could occur in marine symbiotic invertebrates through host shift and specialization. It appears that, as in their terrestrial counterparts, host–symbiont radiations in the marine realm are more prevalent than we expected and thus warrant further investigation.

Genetic differentiation, hybridization and adaptive divergence in two subspecies of the acorn barnacle Tetraclita japonica in the northwestern Pacific

Two acorn barnacles, Tetraclita japonica japonica and Tetraclita japonica formosana, have been recently reclassified as two subspecies, because they are morphologically similar and genetically indistinguishable in mitochondrial DNA sequences. The two barnacles are distinguishable by parietes colour and exhibit parapatric distributions, coexisting in Japan, where T. j. formosana is very low in abundance. Here we investigated the genetic differentiation between the subspecies using 209 polymorphic amplified fragment length polymorphism markers and 341 individuals from 12 locations. The subspecies are genetically highly differentiated (ΦCT = 0.267). Bayesian analysis and principal component analysis indicate the presence of hybrids in T. j. formosana samples from Japan. Strong differentiation between the northern and southern populations of T. j. japonica was revealed, and a break between Taiwan and Okinawa was also found in T. j. formosana. The differentiation between the two taxa at individual loci does not deviate from neutral expectation, suggesting that the oceanographic pattern which restricts larval dispersal is a more important factor than divergent selection in maintaining genetic and phenotypic differentiation. The T. j. formosana in Japan are probably recent migrants from Okinawa, and their presence in Japan may represent a poleward range shift driven by global warming. This promotes hybridization and might lead to a breakdown of the boundary between the subspecies. However, both local adaptation and larval dispersal are crucial in determining the population structure within each subspecies. Our study provides new insights into the interplay of local adaptation and dispersal in determining the distribution and genetic structure of intertidal biota and the biogeography of the northwestern Pacific.

Antennular sensory organs in cyprids of balanomorphan cirripedes: standardizing terminology using Megabalanus rosa

Cirripedes are one of the major groups of fouling organism in the marine environment. The cyprid can, before a permanent attachment, actively explore and walk on the substratum using its antennules in a bipedal fashion without leaving the surface. Studying the structure of the cyprid antennule is therefore important for understanding the events that culminate in biofouling by barnacles. There are at present no complete, standardised accounts of the structure of the cyprid antennules in thoracican barnacles, and moreover, the existing accounts vary in their use of terminology. This article describes the cyprid antennule of the barnacle Megabalanus rosa. This barnacle species is common in E Asia, and the cyprids have previously been used in several biofouling studies. All externally visible setae on the antennules have been mapped; these comprise both chemosensors with a terminal pore, a putative aesthetasc-like seta and mechano-sensory setae. More setae were found on the attachment disc than in previous scanning electron microscope-based studies, but not all structures that can be seen with transmission electron microscopy were visible. The disc itself seems to have a variable surface area, which could assist in exploring rough surfaces. The various lengths of the antennular setae, coupled with the disposition of the segments, enable the cyprid to cover a wide swath of substratum during exploratory walking. A new terminology is proposed for cyprid antennular setae, which will form a basis for future comparative and functional studies of cirripede settlement.

Larval development of Chthamalus malayensis (Cirripedia: Thoracica) reared in the laboratory

Larvae of Chthamalus malayensis (Cirripedia: Thoracica) from Hong Kong were cultured in the laboratory. Larval development includes six naupliar stages and a non-feeding cypris stage. Larvae reached the cypris stage in 20 d at ∼21°C compared to 14 d at ∼28°C. Morphological features including the cephalic shield, frontal horns, labrum, abdominal process, antennules, antennae and mandibles in all nauplius and cypris stages were described and illustrated using a combination of light microscopy and scanning electron microscopy. Attempts were made to compare morphological differences between the nauplii and cyprid of C. malayensis with those of other Chthamalus species including C. stellatus , C. montagui , C. dentatus , C. fragilis , C. dalli , C. antennatus , C. fissus and C. challengeri . The present description of the nauplii of C. malayensis is not in agreement with the previous description of this species.

STUDIES ON TETRACLITA SQUAMOSA AND TETRACLITA JAPONICA (CIRRIPEDIA: THORACICA) II: LARVAL MORPHOLOGY AND DEVELOPMENT

Abstract Nauplius larvae (Stages I–VI) and cyprids of Tetraclita squamosa (Brugiére, 1789) and Tetraclita japonica (Pilsbry, 1916) were cultured and their morphologies compared using light microscopy and scanning electron microscopy (SEM). Using a mixture of Skeletonema costatum Greville, 1866, Isochrysis galbana Parke, 1938, and Tetraselmis Stein, 1878, sp. as food, the larvae of both species completed naupliar development and metamorphosed to cyprids in ∼14 days at 20°C. The body shape and size of the larvae of both species are similar. The major diagnostic morphological difference is the setation of the antennulae and mandibulae. From Stage V to VI, the antennulae of T. japonica bear one more preaxial seta than T. squamosa. From Stage IV to VI, the mandibulae of T. japonica also bear one more simple seta on the endopodite than T. squamosa. The size, shape, antennular morphology, and surface sculpturing of the cyprids of the two species are similar when observed under SEM. The morphology of T. squamosa and of T. japonica is compared with those of the larvae of other Tetraclita species, and taxonomic relationships within the possibly monophyletic Tetraclitidae are discussed.

Metamorphosis in Balanomorphan, Pedunculated, and Parasitic Barnacles: A Video-Based Analysis

Cypris metamorphosis was followed using video microscopy in four species of cirripeds representing the suspension-feeding pedunculated and sessile Thoracica and the parasitic Rhizocephala. Cirripede metamorphosis involves one or more highly complex molts that mark the change from a free cypris larva to an attached suspension feeder (Thoracica) or an endoparasite (Rhizocephala). The cyprids and juveniles are so different in morphology that they are functionally incompatible. The drastic reorganization of the body implicated in the process can therefore only commence after the cyprid has irreversibly cemented itself to a substratum. In both Megabalanus rosa and Lepas, the settled cyprid first passes through a quiescent period of tissue reorganization, in which the body is raised into a position vertical to the substratum. In Lepas, this is followed by extension of the peduncle. In both Lepas and M. rosa, the juvenile must free itself from the cypris cuticle by an active process before it can extend the cirri for suspension feeding. In M. rosa, the juvenile performs intensely pulsating movements that result in shedding of the cypris carapace ∼8 h after settlement. Lepas sp. sheds the cypris cuticle ∼2 days after settlement due to contractile movements of the peduncle. In Lepas anserifera, the juvenile actively breaks through the cypris carapace, which can thereafter remain for several days without impeding cirral feeding. Formation of the shell plates begins after 1-2 days under the cyprid carapace in Lepas. In M. rosa, the free juvenile retains its very thin cuticle and flexible shape for some time, and shell plates do not appear until sometime after shedding of the cypris cuticles. In Sacculina carcini, the cypris settles at the base of a seta on the host crab and remains quiescent and aligned at an angle of ∼60° to the crab’s cuticle. The metamorphosis involves two molts, resulting in the formation of an elongated kentrogon stage with a hollow injection stylet. Due to the orientation of the cyprid, the stylet points directly towards the base of the crab’s seta. Approximately 60 h after settlement the stylet penetrates down one of the cyprid antennules and into the crab. Almost immediately afterwards the unsegmented vermigon stage, preformed in the kentrogon, passes down through the hollow stylet and into the crab’s hemocoel in a process lasting only 30 s. In S. carcini, the carapace can remain around the metamorphosing individual without impeding the process.

Influence of environmental factors on the abundance of Anopheles farauti larvae in large brackish water streams in Northern Guadalcanal, Solomon Islands

BackgroundThe main vector of malaria in Solomon Islands is Anopheles farauti, which has a mainly coastal distribution. In Northern Guadalcanal, Solomon Islands, high densities of An. farauti are supported by large brackish streams, which in the dry season are dammed by localized sand migration. The factors controlling the high larval productivity of these breeding sites have not been identified. Accordingly the influence of environmental factors on the presence and density of An. farauti larvae was assessed in three large naturally dammed streams.MethodsLarval sites were mapped and anopheline larvae were collected monthly for 12 months (July 2007 to June 2008) from three streams using standard dippers. Larval collections were made from 10 locations spaced at 50 m intervals along the edge of each stream starting from the coast. At each collection point, floating filamentous algae, aquatic emergent plants, sun exposure, and salinity were measured. These environmental parameters along with rainfall were correlated with larval presence and density.ResultsThe presence and abundance of An. farauti larvae varied between streams and was influenced by the month of collection, and distance from the ocean (p < 0.001). Larvae were more frequently present and more abundant within 50 m of the ocean during the dry season when the streams were dammed. The presence and density of larvae were positively associated with aquatic emergent plants (presence: p = 0.049; density: p = 0.001). Although filamentous algae did not influence the presence of larvae, this factor did significantly influence the density of larvae (p < 0.001). Rainfall for the month prior to sampling was negatively associated with both larval presence and abundance (p < 0.001), as high rainfall flushed larvae from the streams. Salinity significantly influenced both the presence (p = 0.002) and density (p = 0.014) of larvae, with larvae being most present and abundant in brackish water at < 10‰ seawater.ConclusionThis study has demonstrated that the presence and abundance An. farauti larvae are influenced by environmental factors within the large streams. Understanding these parameters will allow for targeted cost effective implementation of source reduction and larviciding to support the frontline malaria control measures i.e. indoor residual spraying (IRS) and distribution of long-lasting insecticidal nets (LLINs).

CIRRAL LENGTH OF THE ACORN BARNACLE TETRACLITA JAPONICA (CIRRIPEDIA: BALANOMORPHA) IN HONG KONG: EFFECT OF WAVE EXPOSURE AND TIDAL HEIGHT

Abstract Tetraclita japonica is a common barnacle in Hong Kong, occupying a wide vertical zonation on exposed to semi-exposed shores. The length of the exopodite of cirri IV–VI of T. japonica varied with different degrees of wave exposure and tidal levels. Barnacles on exposed shores had shorter cirri than those on semi-exposed ones. Barnacles at the low intertidal also had shorter cirri than those at the high intertidal zone. Differences in desiccation and heat stress, the length of the immersion period for feeding, and predation pressure along the tidal gradient may be additional factors affecting the cirral length of T. japonica. Compared with Balanus glandula in temperate waters, the proportional difference in the cirral length of T. japonica between wave-extreme locations is much smaller (4%) than that of B. glandula (80%). Differing from B. glandula, which actively beat their cirri to feed, T. japonica exhibits prolonged extension of the cirri for feeding, which may not favour long cirri even on semi-exposed shores. Barnacles with different cirral activities, therefore, appear to have different degrees of cirral responses to environmental stimulus.

Chemistry and biology of maculalactone A from the marine cyanobacterium Kyrtuthrix maculans

Maculalactone A is the most abundant secondary metabolite in Kyrtuthrix maculans, a marine cyanobacterium found in the mid-high shore of moderately exposed to sheltered rocky shores in Hong Kong and South East Asia. This species appears to survive as ‘pure’ colonies forming distinct black zones on the rock. Maculalactone A may provide K. maculans with a chemical defense against several marine organisms, including the common grazer, Chlorostoma argyrostoma and settlement by larvae of the barnacles, Tetraclita japonica, Balanus amphitrite and Ibla cumingii. The natural concentration of maculalactone A varied with season and also with tidal height on the shore and although a strong positive linear correlation was observed between maculalactone A concentration and herbivore grazing pressure, manipulative experiments demonstrated that grazing pressure was not directly responsible for inducing the biosynthesis of this metabolite. The potential of maculalactone A as a ‘natural’ marine anti-fouling agent (i.e. as an alternative to environmentally-damaging copper- and tin-based anti-fouling paints) was investigated after achieving a gram-scale synthesis of this compound. Preliminary field trials with anti-fouling paints which contained synthetic maculalactone A as the active principle have confirmed that this compound seems to have a specific activity against molluscan settlers.